Enterohemorrhagic Escherichia coli (EHEC) are now recognized as the causative agents of enterohemorrhagic colitis (EC), hemolytic uremic syndrome (HUS) and mesenteric adenitis (MA). Enterohemorrhagic colitis is characterized by severe, crampy abdominal pain, initially watery diarrhea followed by copious, bloody diarrhea, with little or no fever; hemolytic uremic syndrome is one of the complications resulting from enterohemorrhagic colitis. Enterohemorrhagic colitis occurs most frequently in developed countries, and most outbreaks of this disease have been associated with the consumption of contaminated meats (e.g., undercooked ground beef) and dairy products (e.g., raw milk). Escherichia coli serotype O157:H7 is the most frequent EHEC isolate, but many other serotypes of E. coli that are capable of causing equally devastating foodborne outbreaks of EC and HUS have been identified. Presently, the EHECs and, in particular, E. coli O157:H7 are among the most serious bacterial pathogens confronting the public health and food safety agencies.
To date, however, there is no known single genetic marker for virulent EHEC strains which can be used to detect not only O157:H7, but all serogroups of EHEC. Current immunological assays based on the O157:H7 serogroup generate false negatives due to the existence of many non-O157:H7 serogroups of EHEC. Moreover, immunological assays require a lengthy microbiological work-up and, thus, are expensive and time-consuming. In contrast to immunological assays, current rapid, genetic methods target the genes that encode the shiga-like toxins I and II (i.e., slt-I and slt-II respectively) and a particular allele of uidA. The presence of these genes is broadly associated with EHEC, but no real correlation with virulence can be established.
In fact, there are known EHECs positive for both slt-I and slt-II, but there are also known EHECs that are negative for both slt-I and slt-II or, that are positive for only one slt. Moreover, many bacteria possess slt-I and/or slt-II genes without being associated with EHEC, HUS or EC. Enteropathogenic E. coli (EPEC), for example, is slt-positive, but causes only mild, persistent diarrhea. In addition, an entire new class of diarrheagenic E. coli, known as entero-SLT-producing and invasive E. coli (ESIEC), has been identified that is positive for sit. Of 54 strains belonging to ESIEC, 53 were slt-I positive and 3 were slt-II positive. Furthermore, Shigella dysenteriae type I possesses slt-I, but causes a very different disease. As such, slt probes detect entire classes of bacteria irrelevant to EHEC and, thus, the presence of slt-I and/or slt-II genes is not an adequate indicator of EHEC risk.
Further, the uidA allele identifies many O157:H7 strains of EHEC. It is recognized, however, that uidA does not encode a real virulence factor. The uidA gene is not expressed in EHEC because the allele characteristic of O157:H7 is not an open reading frame and, thus, it is unable to express a protein. It is believed that this allele is most probably a residual trait of the ancestral EHEC from which the O157:H7 serogroup clone evolved. In addition, the uidA gene is not real useful as a target because it does not detect non-O157:H7 EHEC strains. Although E. coli O157:H7 is the major serogroup of EHEC, other serogroups of E. coli which can cause EC and HUS cannot be ignored. E. coli strains O157:H45, O157:H19, O26:H11, O22:H8, O55:H6, O11:H8, O11:H- and O145:NM, for example, are also isolated from victims of EC and HUS. Moreover, it is likely that more E. coli serotypes will be identified as EHEC agents when the genetic mechanisms underlying the pathogenesis of this organism is more fully understood.
From the foregoing, it is apparent that the immunological assays presently being used to detect EHECs fail to detect non-O157:H7 EHECs and, in addition, they are labor-intensive and time-consuming. Moreover, the rapid, genetic assays being used are founded on known genes that do not correlate well with EHEC and, thus, they are prone to false positives and false negatives. False positives result from the relatively harmless slt-bearing bacteria; false negatives result from the many EHEC strains lacking slt-I, slt-II and uidA, all of which can cause devastating EC and HUS. As such, there still remains a need in the art for rapid, sensitive and highly specific methods for the detection of enterohemorrhagic E. coli and, in particular, O157:H7 E. coli. The present invention remedies this need by providing such methods.